SUMMARY ADP-ribosylation is a widespread and ubiquitous post-translational modification across all kingdoms of life. Although it was the first PTM described, the biochemical selectivity and cellular consequences of ADP- ribosylation is very poorly understood. The objective of this project is to define the pathways linking unique ADP- ribosylation modifications to their downstream function in the cell. We will use this information to develop first-in- class peptide-based inhibitors to target malfunctioning ADP-ribose signaling networks. In humans, 17 different poly-ADP-ribose polymerases (PARPs) catalyze ADP-ribosylation. PARP enzymes have been implicated in a number of physiological functions (e.g. DNA repair, RNA regulation, cell development) and a broad array of diseases, most notably viral infection. While previous efforts have validated the roles for PARPs 13, 14, and 15 in viral regulation, their mechanism of action remains unclear. In large part, this lack of clarity arises from our inability to connect a single site of ADP-ribosylation to its downstream function. Work in the kinase field has demonstrated the power of connecting enzyme-specific post-translational modification sites (PTMs) to their specific cellular function, fundamentally altering our understanding of signal transduction and revealing new avenues for therapeutic intervention in disease. By leveraging the expertise from my lab and that of my collaborators, our work will use a multidisciplinary approach to determine the functional consequences of unique ADP-ribosylation events in the cell and develop novel PARP-selective peptide-based inhibitors to block specific modifications. In the first aim, we will define the sites of modification for both PARP14 and PARP15 on themselves (auto-modification) and on other targets (trans-modification). We have worked extensively with these PARP family members to define their specific protein targets and we have a robust viral model to interrogate how specific site alterations change pathway behavior. Our work in this aim will provide the first links between specific ADP-ribosylation sites and their effects in the cell and it will be adaptable to the other PARPs. In the second aim, a PARP14-specific target peptide sequence that we have developed will serve as a platform to identify selective PARP14 inhibitors. We will synthesize peptide and peptoid (poly-N-substituted glycine) derivatives of the initial target peptide and identify structure activity relationships (SAR analysis) with the resulting library. This work will expand our knowledge of the biochemical mechanisms governing target selection and will provide a novel avenue for targeting PARPs to treat disease. The work from this proposal will be broadly applicable to the study of the remaining PARP family members and will have applications in medicine, cell biology, developmental biology, and the study of the post-translationally modified proteome.